Autoimmune diseases result from the failure of the immune system to develop tolerance to self-proteins. The signaling threshold of antigen receptors and costimulatory receptors determine immunity or tolerance to self-proteins. A major costimulatory receptor, CD28, is required for induction of autoimmunity in several mouse models. CD28 costimulation amplifies early and late T cell receptor (TCR)-mediated signaling events. It has been shown that blocking CD28-mediated costimulation may represent one potential immunotherapy against autoimmunity. However, the molecular mechanisms that maintain immune tolerance to self-antigen in vivo and integrate costimulatory signals with the TCR signals are poorly understood. Recent studies suggest that CD28 costimulation may lower the threshold needed for T cell activation. Casitas-B-lineage lymphoma-b protein (Cbl-b), an adaptor protein and E3 ubiquitin ligase, can control CD28-dependent T cell activation via the regulation of Vav activity, suggesting a critical role of Cbl-b in the regulation of T cell activation threshold and hence in the maintenance of a balance between immunity and tolerance. Mice lacking Cbl-b spontaneously develop autoimmunity or have an increased susceptibility to experimental allergic encephalomyelitis. Therefore, Cbl-b may set the threshold for T cell activation. Our preliminary experiments showed that stimulation with higher doses of anti-CD3 partially overcomes defective T cell proliferation in CD28-deficient mice. Moreover, the presence or absence of CD28 may control TCR-induced Cbl-b ubiquitination and degradation. These findings provide a novel molecular mechanism(s) for how CD28 costimulation mediates optimal T cell activation. Based upon the above data, we hypothesize that Cbl-b regulates CD28-mediated T cell activation. Specifically, we will investigate whether and how CD28costimulation controls ubiquitination of Cbl-b; whether Cbl-b regulates CD28-mediated formation of immunological synapse; and whether Cbl-b regulates CD28-dependent autoreactive T cell activation in autoimmune arthritis. The information generated by the proposed studies will lead to a better understanding of Cbl-b in T cell biology and in autoreactive T cell responses, and will shed light on the development of novel therapeutic approaches to autoimmune arthritis.